In the United States, human papillomaviruses (HPVs) cause an estimated 6 million new infections annually. Some HPVs cause the most prevalent benign viral sexually transmitted hyperproliferative disease, genital warts, and are referred to as low risk (LR) HPVs (e.g., HPV 6). Others cause cervical cancer and some head and neck cancers and are, therefore, referred to as high risk (HR) HPVs (e.g., HPV 16). All HPVs are maintained as low copy number extrachromosomal genomes in the undifferentiated layer and amplify their genomes and complete their life cycles in the differentiated compartment of the epithelium where cells normally have exited the cell cycle. All HPVs have to create an S-phase environment in this latter compartment to complete their life cycle. While the HR HPVs have been extensively studied because of their ability to cause human cancer, it is imperative that we understand the mechanism of pathogenicity of the LR HPVs, clinically significant pathogens in their own right. We have been focusing on the E7 proteins encoded by LR and HR HPVs because of their pivotal role in both the life cycle and oncogenesis. This application builds on our novel observation that both LR and HR HPVs can target the pRb family member, p130/pRB2, for degradation while only HR HPVs can target other members of the pRb family. Previously, it was thought that only HR HPV E7 could target proteins for degradation. Building on our paradigm-shifting observation, the Specific Aims of this exploratory R21 application are: 1) to test the hypothesis that LR and HR HPV E7 target other (non-pRb family member) proteins for degradation and 2) to test the hypothesis that LR HPV E7 binds members of the ubiquitin ligase complex. We will use mass spectrometry to detect endogenous proteins that are differentially ubiquitylated in the presence of LR HPV 6 and/or HR HPV 16 E7. We will identify proteins that form a complex with LR HPV 6 E7 by tandem affinity purification coupled with LC MS/MS. Results will be appropriately validated. By generating the first global comparison of the substrates ubiquitylated in response to LR and/or HR E7 expression, a profile of proteins that contribute to the viral life cycle versus those involved in oncogenesis will be developed. The analysis of HPV 6 E7 binding partners, by comparison to HPV 16 E7 binding partners, will identify ubiquitin ligases that potentially mediate LR HPV 6 E7 and/or HR HPV 16 E7 targeted degradation, providing possible novel therapeutic targets for directed inhibition of such degradation. By identifying shared E7 activities in Specific Aims 1 and 2 we will gain new insight into potential biomarkers of and therapeutic targets pertinent to the life cycle of all HPVs. Subtracting out these shared activities will identify biomarkers and therapeutic targets specific to the oncogenic activity of the HR HPVs. The feasibility of these experiments is high based on data in the literature and the expertise of the two laboratories involved in their execution. PUBLIC HEALTH RELEVANCE: Human papillomaviruses, as causative agents of cervical cancer and some head and neck cancers, have a major public health impact. Although the FDA-approved HPV vaccine holds promise of preventing infection, it cannot cure existing infections. This application offers the potential of identifying new biomarkers for diagnosis and monitoring of disease status as well as potential therapeutic targets, and thereby the translation of the results from the bench to the bedside.